Strategy to increase anti-viral, anti-microbial, and anti-fungal defense

ABSTRACT

Disclosed herein are compositions and methods for inducing, promoting, or enhancing an immune response in a subject. For example, the disclosed compositions and methods can be used prophylactically to prevent viral/microbial infections or therapeutically to treat acute infections. In some embodiments, the disclosed method involves administering to the subject a composition comprising in vitro transcribed (IVT) RNA comprising short interspersed nuclear elements (SINEs), such as Alu repeats. In some embodiments, the disclosed compositions and methods can be used to induce, promote, or enhance any immune response in a subject, including an anti-viral, anti-microbial, anti-fungal, or anti-parasite response. In some embodiments, the disclosed compositions can be administered to any mucosal barrier, such as lungs or intestines, e.g. to enhance an innate immune response against a virus or pathogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/408,518, filed May 10, 2019, which claims priority to U.S.Provisional Application No. 62/669,747, filed May 10, 2018, both ofwhich are hereby incorporated herein by reference in their entireties.

BACKGROUND

More than 50% of the human genome is composed of repetitive elements, ofwhich Alu repeats (Alus) are the most abundant and comprise 11% of thegenomic sequence. Alus, named after the internal Alu/restriction sitefound in these repeats, belong to the short interspersed nuclearelements (SINEs) and are evolutionary derived from the 7SL RNA gene.Alus are expressed in germ cells and embryonic pluripotent stem cellsbut are epigenetically silenced by DNA methylation as the cellsdifferentiate. Alterations of this epigenetic control is implicated inwide range of disorders including autoimmune diseases (Hung, T. et al.,Science 350:455-459 (2015)), macular degeneration (Kaneko H. et al.,Nature 471:325-330 (2011)), neurologic and neurodegenerative disorders(Baillie, J. K. et al., Nature 479:534-537 (2011)).

Alus contain an internal promoter and can be transcribed independentlyby RNA polymerase III in response to various stress conditions includingviral infection, heat shock, translational inhibition or DNAmethyltransferase inhibitors (Liu, W. M. et al., Nucleic Acids Res23:1758-1765 (1995); Chu, W. M. et al., Mol Cell Biol 18:58-68 (1998)).These independent Alu transcripts are ˜300 bp in length and are composedof two monomers separate by an A-rich sequence. Alus can also beembedded within mRNA or long non-coding RNA and transcribed by RNApolymerase II. These embedded Alus are expressed at relatively higherlevels and can be full or partial length with one or more copies in thesense or antisense orientation that form intermolecular base-pairing andsecondary structures.

Sequencing of the human genome has revealed that Alus are underevolutionary selection as they are distributed non-normally throughoutthe genome, with the highest Alu density found in chromosome 19 (Lander,E. S. et al., Nature 409:860-921 (2001)). However, the beneficialcontribution of Alus to human hosts is still largely unknown.

SUMMARY

Retrotransposons comprise approximately 45% of human genome of whichapproximately 11% comprise short interspersed elements (SINEs). Asdisclosed herein, sense and/or antisense SINEs can increase ininterferon type III which is known to have antiviral/antimicrobialactivity. In addition, sense and/or antisense SINEs can induce innateimmune response. Moreover, sense and/or antisense SINEs may form siRNAthat target viral, bacterial, fungi and parasite genome for degradationand/or inhibit translation/replication.

Therefore, disclosed herein are compositions and methods for inducing,promoting, or enhancing an immune response in a subject. For example,the disclosed compositions and methods can be used prophylactically toprevent viral/microbial infections. In some embodiments, the disclosedmethod involves administering to the subject a composition comprising invitro transcribed (IVT) RNA comprising short interspersed nuclearelements (SINEs). In some embodiments, the disclosed compositions andmethods can be used to induce, promote, or enhance any immune responsein a subject, including an anti-viral, anti-microbial, anti-fungal, oranti-parasite response. In some embodiments, the disclosed compositionscan be administered to any mucosal barrier, such as lungs or intestines,e.g. to enhance an innate immune response against a virus or pathogen.

In some embodiments, the disclosed compositions and methods induceinterferon lambda 2 (INFL2), interferon lambda 3 (INFL3), and downstreamISGs. In some embodiments, the disclosed compositions are used incombination with viral antigens to enhance viral immunity.

In some embodiments, the SINE comprises Alu repeats. Alu elements aresplit into subfamilies, such as AluJ, AluS, and AluY. The dominant Ssubfamilies included Sx, Sq, Sp and Sc. Ya5 and Yb8 are the dominant Ysubfamilies in humans.

Modern Alu elements are about 300 base pairs long and are thereforeclassified as short interspersed nuclear elements (SINEs). In someembodiments, the composition comprises the full length Alu element.However, fragments of the Alu element can also be used includingfragments at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 141, 142, 143, 144, 145, 156, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, or 300bp in length. The composition can contain sense strands, antisensestrands, or a combination thereof. The composition can also comprisescombinations of different Alu elements in the same or differentorientations.

In some embodiments, the IVT RNA sense strands, antisense strands, orcombinations thereof are modified nucleotides to reduce innate immuneresponses. For example, the IVT RNA strands can comprises modifiednucleosides, such as pseudouridine (abbreviated by the Greek letter“psi” or NJ″), N1-methylpseudouridine, 5-methylcytosine (m5C),5-methyluridine (m5U), 2′-O-methyluridine (Um or m2′-OU), 2-thiouridine(s2U), or N6-methyladenosine (m6A)).

Also disclosed herein is a vaccine composition that contains an in vitrotranscribed (IVT) RNA comprising short interspersed nuclear elements(SINEs) as disclosed herein, in a pharmaceutically acceptable carrier.The disclosed vaccine composition can in some embodiments, contain oneor more antigens, such as viral antigens. The disclosed vaccinecomposition can in some embodiments, contain an adjuvant.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A to 1L. Activation of C19MC by induces resident miRNA andcellular defense genes. FIG. 1A. Arrangement of the upstream CpG island,resident miRNA, repetitive elements and the CG content within C19MC.FIGS. 1B and 10 . Venn diagrams of upregulated (FIG. 1B) ordownregulated miRNAs (FIG. 10 ) in AdHEK cells transfected with759-sgRNA/SAM, 620-sgRNA/SAM or GFP transfected Control for 72 hrfollowed by sRNAseq. Resident miRNA of C19MC are shown in magenta. FIG.1D. Gene set enrichment analysis performed on differentially expressedmRNAs in 759-sgRNA/SAM and 620-sgRNA/SAM compared to GFP control. FIGS.1E and 1F. RT-PCR for hsa-miR-517a normalized to U18 (FIG. 1E) orIFNL2/3 normalized to GAPDH (FIG. 1F) in AdHEK cells transfected with759-sgRNA/SAM or GFP Control for 72 hr. FIGS. 1G and 1H. RT-PCR for theindicated gene normalized to GAPDH in untreated AdHEK293 cells (FIG. 1G)or treated with the indicated amount of IFNL1/3 for 48 hr (FIG. 1H).FIGS. 1I-1L. RT-PCR for hsa-miR-517a normalized to U18 (FIGS. 11 and 1K)or IFNL2/3 normalized to GAPDH (FIGS. 1J and 1L) in the indicated cellstransfected with 759-sgRNA/SAM or GFP control. Data represent mean±SEMof a representative experiment of at least three independent experimentsperformed in triplicate. *p<0.05 versus GFP.

FIGS. 2A to 2D. Induction of IFNL2/3 by transcriptional activation ofC19MC is independent of miRNA activity. FIG. 2A. RepresentativeImmunoblot for DICER1, TUBA1B and GAPDH in HEK293T or NoDICE 2-20 cells(FIGS. 1B and 1C) RT-PCR for miR-517a normalized to U18 (FIG. 2B) orIFNL2/3 normalized to GAPDH (FIG. 2C) in the indicated cells transfectedwith 759-sgRNA/SAM or BB-sgRNA/SAM for 72 hr. FIG. 2D. IFNL3 proteinlevel in the conditioned media of HEK293T or NoDICE 2-20 cellstransfected with 759-sgRNA/SAM or BB-sgRNA/SAM for 72 hr. Data representthe mean±SEM of a representative experiment of at least threeindependent experiments performed in triplicate. *p<0.05 versusBB-sgRNA/SAM.

FIGS. 3A to 3F. IFNL2/3 expression after C19MC activation is driven byAlu RNA production. FIG. 3A. Representative Primer extension to assayfor the abundances of Alu RNA in HEK293T and NoDICE 2-20 cellstransfected with 759-sgRNA/SAM or BB-sgRNA/SAM for 72 hr. FIG. 3B.Arrangement of the CYP19A1 gene with the repetitive elements and the CGcontent. FIGS. 3C and 3D. RT-PCT for CYP19A1 normalized to GAPDH (FIG.3C) and IFNL2/3 normalized to GAPDH (FIG. 3D) in HEK293T cellstransfected with 759-sgRNA/SAM (759), 47.2-sgRNA/SAM (47.2),125.3-sgRNA/SAM (125.3) or BB-sgRNA/SAM (BB) for 72 h. FIGS. 3E-3F.RT-PCR for IFNL2/3 normalized to GAPDH in HEK293T cells (FIG. 3E) orHTR8/SVneo cells (FIG. 3F) transfected for 18 h with dsDNA, in vitrotranscribed GFP mRNA or the indicated forward (FIG. 3F) reverse (R) orboth RNA fragments. Data represent the mean±SEM of a representativeexperiment of three independent experiments performed in triplicate.*p<0.05 versus BB-sgRNA/SAM (FIGS. 3C and 3D); versus non transfectedcontrol cells (NTC, FIGS. 3E and 3F).

FIGS. 4A to 4E. Colocalization of C19MC and Alu RNA in human andC19MC-transgenic mouse placentas (FIGS. 4A-4B) RT-PCT of hsa-miR-517anormalized to U18 (FIG. 4A) or representative agarose gel of IFNL2/3 andGAPDH (FIG. 4B) in iPSC and differentiated normal human fibroblast(NHDF). FIGS. 4C-4E. In situ hybridization for Alu RNA in human termplacenta pretreated with or without RNaseA prior to probing (FIG. 4C);Serial sections of first trimester human placentas stained forCytokeratin (fetal) and Vimentin (maternal decidual cells) (FIG. 4D); orplacental sections of VVT and C19MC-transgenic mice (FIG. 4E).miR-517a/c (purple) Alu RNA (purple) and Nuclei was counterstained inred (FIGS. 4C-4E). Scale bars represent 100 μm in full image and 50 μmin insets; original magnification 10×.

FIG. 5 . Induction of Alu expression by cellular stress activatesIFNL2/3 expression. RT-PCR for IFNL2/3 normalized to GAPDH in HeLa cellstreated with 100 μg/ml cyclohexamide for 4 hr, or heat shock for 30 minat 45° C. and allowed to recover for 2 hr prior to collection of totalRNA.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, biology, and the like, which arewithin the skill of the art.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the probes disclosed and claimed herein.Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C., and pressure is at or near atmospheric. Standardtemperature and pressure are defined as 20° C. and 1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “sample from a subject” refers to a tissue (e.g., tissuebiopsy), organ, cell (including a cell maintained in culture), celllysate (or lysate fraction), biomolecule derived from a cell or cellularmaterial (e.g. a polypeptide or nucleic acid), or body fluid from asubject. Non-limiting examples of body fluids include blood, urine,plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitialfluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid,saliva, anal and vaginal secretions, perspiration, semen, transudate,exudate, and synovial fluid.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

Disclosed herein are in vitro transcribed (IVT) RNA comprising shortinterspersed nuclear elements (SINEs). In some embodiments, Alu RNAtemplates are generated by PCR using primers that have T7 promoter inthe forward or reverse strand to allow in vitro transcription of thesense or antisense strands. In vitro transcription of the Alu templatescan be carried out using HiScribe T7 High Yield RNA synthesis kit (NEB)with partial of complete nucleotides substitution with modifiednucleotides.

Also disclosed herein is a vaccine composition that contains an in vitrotranscribed (IVT) RNA comprising short interspersed nuclear elements(SINEs) as disclosed herein, in a pharmaceutically acceptable carrier.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1: Increase in Alu Transcripts Activates CellularDefense Response by Inducing Interferon Type III

Methods

Ethical Approvals

Placentas from voluntary terminations of uncomplicated first trimester(7-8 weeks) and early (20 weeks) pregnancies were obtained with written,informed patient consent. This work was carried out with approval fromthe University of South Florida Institutional Review Board (protocol#00015578). The generation, care, and use of the C19MC humanized micewas carried out at University of Pittsburgh with approval from theInstitutional Animal Care and Use Committee¹.

Cell Culture

AD-293 cells (Stratagene 240085), 293T cells (ATCC CRL-3216), NoDice2-20 cells were a gift from Dr. Bryan Cullen² and B16-F10 cells (ATCCCRL-6475) were grown in DMEM supplemented with 10% heat inactivatedfetal bovine serum (Millipore-Sigma). HTR8/SVneo (ATCC CRL-3271) cellswere cultured in RPMI supplemented with 5% heat inactivated fetal bovineserum. Normal human dermal fibroblasts (NHDF) (Lonza CC-2511) werecultured in Fibrolife Serum-free medium (Lifeline Cell Technology).iPSCs (ATCC ACS-1030) were cultured in Essential 8 medium(ThermoFisher).

For IFNL3 stimulation AD-293 cells were seeded in multi-well plates at adensity of 10⁵ cells/mL 24 hours prior to addition of recombinant IFNL3protein (PBL Assay Science #11730-1) to the medium. After 48 hours, theexpression of interferon response genes ISG15 and OAS1 were assessed byRT-PCR.

Transient Activation of C19MC and CYP19A1 Using Cas9-SAM

gRNAs were designed to C19MC using the ATUM CRISPR gRNA Design tool toidentify PAM sequences near the first miRNA of the cluster. gRNAs forCYP19A1 were designed. Adapter sequences were added to the gRNA prior tooligo synthesis (Oligos #1-8) to allow for Golden Gate cloning into thelenti ssgRNA(MS2)-zeo-backbone (Addgene #61427) and subsequenttransformation in to GC10 competent cells. Ampicillin resistant cloneswere selected and verified by sequencing.

AD-293, 293T, 2-20, or HTR8 cells were seeded in multi-well plates 24hours before transfection with 1:1:1 mass ratio of ssgRNA(MS2)-zeo,MS2-P65-HSF1 and dCas9-VP64-GFP plasmids (Addgene #61426 and 61422,respectively) using Lipofectamine 2000 (Invitrogen). Cell culture mediumwas changed after 24 hours.

miRNA and mRNA Sequencing

Total RNA was isolated from HEK293 cells 72 hours after transfectionwith 759-sgRNA/SAM or 620-sgRNA/SAM using the miRNeasy Kit (QIAGEN) andstored at −80° C. in RNAse-free water.

Two micrograms of total RNA were converted into a small RNA cDNA libraryas previously described⁴. RNA inputs for each sample were ligated to abarcoded 3′ adapter sequence, pooled, size selected, and gel purifiedbefore ligation of 5′ adapters. The RNA was again size selected and gelpurified before second strand synthesis using SuperScript III, alkalineRNA hydrolysis, and 10 cycles of PCR amplification.

Individual RNAseq libraries were quality controlled using an AgilentTapeStation with High Sensitivity D1000 ScreenTape. Indexed samples werequantified using the Qubit dsDNA HS assay and pooled at equimolarconcentrations (10 nM). Libraries were sequenced on an Illumina NextSeq500 using 75-bp paired-end reads in mid-output mode.

Bioinformatics Analysis

miRNA sequencing read annotation was performed. Reads that mapped tomore than one location were assigned to each mapping transcript as afraction of the number of mapping locations (fractional mapping). mRNAsequencing reads were aligned to the human genome (GRChg38) using theRNASTAR aligner⁶ allowing for two mismatches. Read counts were generatedusing featureCounts⁷ and differential expression analysis was completedusing edgeR. Differentially expressed genes were considered significantwith an FDR<0.1 and fold-change>2.0 up or down.

ELISA

Enzyme-linked immunosorbance assay for IFNL1/3 (R&D Systems #DY1587) wasperformed according to the manufacturer's instructions on conditionedcell culture media collected 72 hours after 759-sgRNA/SAM transfectionof AD-293, 293T, or 2-20 cells.

In Situ Hybridization

In situ hybridization for hsa-miR-517a/c, as a representative member ofthe C19MC, and Alu repeats in human and mouse placental sections wasperformed according the manufacturer's (Exiqon) instructions. Briefly,paraffin embedded tissue sections were deparaffinized in xylene andrehydrated by a series of graded alcohol washes. In situ hybridizationwas then performed using 40 nM 5′,3′-digoxignein-labeled locked nucleicacids (Exiqon) complementary to has-miR-517a/c (Exiqon #90005), Alu(Oligo #9) or a scrambled control. Hybridization and post-hybridizationSSC washes were performed at 55° C. Sections were then blocked, and thehybridization probes were detected using alkaline phosphatase-conjugatedsheep anti-digoxignenin Fab fragments (Roche #11093274910). Signal wasdeveloped using NBT/BCIP (Roche #11697471001) to produce the dark-bluestaining. Nuclei were counterstained using Nuclear Fast Red (VectorLaboratories #H-3403). Slides were then dried and covered for imageanalysis.

RT-PCR

For mRNA RT-PCR analysis 1 μg total RNA was reverse transcribed usingrandom hexamer or oligo(dT) primers and M-MuLV reverse transcriptase(New England Biolabs #M0253) according to the manufacturers'recommendations. For miRNA cDNA, 0.5 μg total RNA was reversetranscribed using the TaqMan miRNA Reverse Transcription Kit(ThermoFisher Scientific #4366596) according to the manufacturer'sinstructions.

To assess relative mRNA and miRNA expression levels, RT-PCR of the cDNAproducts was performed using the following TaqMan RT-PCR probes: humanIFNL2/3 (Hs04193048_gH), mouse IFNL2/3 (Mm04204158_gH), OAS1(Hs00973637_m1), ISG15 (Hs00192713_m1), IFNLR1(Hs00417120_m1), IL10RB(Hs00175123_m1), CYP19A1 (Hs00903411_m1), human GAPDH (Hs02786624_g1),mouse GAPDH (Mm99999915_g1), hsa-miR-515-5p (001112), hsa-miR-516a-5p(002416), hsa-miR-516b (001150), hsa-miR-517a (002402), hsa-miR-518c(002401), hsa-miR-519d (002403), hsa-miR-379 (001138), hsa-miR-412(001023), hsa-miR-485 (001036), hsa-miR-654 (002239), U18 (001204).TaqMan probes were used according to the manufacturer's instructionswith TaqMan Fast Advanced Master Mix (ThermoFisher #4444556) on aQuantStudio 3 (Life Technologies). Expression of mRNA and miRNA wasnormalized to GAPDH and U18, respectively. Relative expression wascalculated using the 2^(−ΔΔCt) method.

Primer Extension

Oligonucleotides complementary to Alu (Oligo #10) and beta actin (Oligo#11) were 5′-labeled with [γ-³²P]ATP using T4 PNK (New England Biolabs#M0201). The labeled primers were then annealed to 20 μg of total RNAisolated from 293T or 2-20 cells 72 hours after activation of C19MC withsgRNA759/SAM and reverse transcribed with M-MuLV. The resulting DNA wassubjected to gel electrophoresis on an 8% TBE-urea gel. The gel wasdeveloped on a phosphor storage screen (GE Life Sciences) and scannedusing a Amersham Typhoon (GE Life Sciences).

Dot Blotting of dsRNA

1, 2, or 4 μg of total RNA or in vitro transcribed Alu RNA in a 10 μLvolume of TE with 350 mM NaCl was loaded onto a wet BiotraceNitrocellulose membrane (Pall Biosciences) using a Minifold dot-blotter(Schleicher & Schuell, Inc.). The membrane was then baked at 80° C. for1 hour before blocking for 1 hour at room temperature in Odyssey PBSblocking buffer (LI-COR). The membrane was then probed overnight with J2mouse anti-dsRNA antibody (Scicons, 1:2000) followed by a 20 minuteincubation with IRDye 800CW goat anti-mouse secondary antibody (LI-COR,1:2500). The membrane was washed three times in TBS-Tween after eachantibody incubation. Membranes were imaged on an Odyssey CLx (LI-COR)and analyzed using ImageStudio software (LI-COR).

RNaseA digestion of ssRNA was performed in TE with 350 mM NaCl to blockdsRNA digestion. RNaseA was added to the total RNA or in vitrotranscribed Alu RNA to a final concentration of 10 μg/mL and incubatedfor 2 hours at room temperature before dot blotting.

Western Blotting

Protein lysates were fractionated by SDS-PAGE and transferred toBiotrace Nitrocellulose membrane (Pall Biosciences). Membranes wereincubated overnight at 4° C. with rabbit anti-alpha tubulin (CellSignaling Technologies #2144, 1:1000) and rabbit anti-DICER1 (CellSignaling Technologies #5362S, 1:1000) primary antibodies in Odyssey PBSblocking buffer (LI-COR) before a 20 minute room temperature incubationwith IRDye 680 donkey anti-rabbit secondary antibody (LI-COR, 1:5000).Membranes were washed three times in TBS-Tween after each antibodyincubation, imaged on an Odyssey CLx (LI-COR), and analyzed usingImageStudio software (LI-COR).

In Vitro Transcription

Template generation using oligos #12-13, in vitro transcription, cappingand poly(a) tailing of GFP mRNA was performed. Control 350 nt GFP RNAtemplates were generated by PCR of pLL3.7 plasmid (a gift from Dr. LukParijs, Addgene plasmid #11795) using oligos #14-15. A second round ofPCR using oligos #15 and #16 (GFP F) or #14 and #17 (GFP R) wasperformed to add a T7 promoter for transcription of the sense orantisense strands respectively. Alu RNA templates were similarlygenerated by PCR of a bacterial artificial chromosome containing C19MCusing primer pairs specific for AluJb7 (#18 and #19), AluSz8 (#22 and#23), or AluSx10 (#26 and #27). In addition a template containing allthree Alu repeats (Alu3x) was generated using oligos #18 and #27. T7promoters were added to the 5′ end of either strand of the PCR productsduring a second round of PCR (AluJb7 F: #19 and #20; AluJb7 R: #18 and#21; AluSz8 F: #23 and #24; AluSz8 R: #22 and #25; AluSx10 F:#27 and#28; AluSx10 R: #26 and #29; Alu3x F: #20 and #27; Alu3x R: #18 and #29)to allow transcription of the sense or antisense strands.

In vitro transcription of the GFP and Alu templates was carried outusing HiScribe T7 High Yield RNA synthesis kit (NEB) with completesubstitution of uridine for pseudouridine (TriLink Biotechnologies,N-1019). The RNA products were precipitated in 5 M ammonium acetate,resuspended in H₂O, quantified by spectrophotometry, and stored at −80°C. until use.

Statistics

Two-tailed Student's T-test or one-way ANOVA with Tukey's post-hoc testwere used when appropriate for testing statistical significance usingGraphPad Prism 7 software. P<0.05 was considered to be statisticallysignificant unless specified differently in the relevant figure legend.All data are representative of at least three independent experimentsunless otherwise noted in the relevant figure legends.

Results and Discussion

Transcriptional Activation of 100 kb C19MC Cistron by CRISPR/SAM

To investigate the role of Alus in physiological settings, focus was onthe largest human microRNA (miRNA) cistron, mir-498(46) cistron locatedon chromosome 19, known as C19MC. This miRNA cluster spans over 100 kband contains 46 miRNA genes and 265 Alus are embedded in both the senseand antisense strand. C19MC is epigenetically regulated by imprinting.In the placenta, the paternal allele is transcribed by polymerase II asa single RNA transcript (FIG. 1A) (Noguer-Dance, M. et al., Humanmolecular genetics 19:3566-3582 (2010)). To transcriptionally activatethe entire C19MC cistron, the CRISPR/Cas9-SAM (SAM) technology(Konermann, S. et al., Nature 517:583-588 (2015)) was employed using twodifferent single guide RNA (sgRNAs), the 620-sgRNA which binds at ˜579bp upstream of the first miRNA or the 759-sgRNA that binds at ˜171 bpupstream of the first two miRNAs of the cistron. Small RNA sequencing(sRNAseq) analysis of AdHEK cells transiently transfected with759-sgRNA/SAM, 620-sgRNA/SAM or GFP control for 72 hours showed aconsistent increase, up to 8,000-fold, in the expression of 39 miRNAs ofthe C19MC cistron (threshold: >2-fold, FDR 0.10) (FIG. 1B). Five othermiRNAs were upregulated in both 759- and with 620-sgRNA/SAM transfectedcells, however none of them belonged to the C19MC cistron (FIG. 1B). Onthe other hand, only 3 miRNAs were consistently downregulated by 759-and 620-sgRNA/SAM, but none of them belonged to the C19MC cistron (FIG.10 ). The 8 differentially expressed miRNAs (3 downregulated and 5upregulated) in both 759- or 620-sgRNA/SAM-transfected cells may beregulated by the C19MC. Activation of C19MC was confirmed by RT-PCR,demonstrating a 135-fold increase in miR-517a, a member of C19MC, after759-sgRNA/SAM transfection compared to GFP control. These data show thatCRISPR/SAM technology specifically activate the C19MC cistron withminimal off target effects.

C19MC Induces Interferon and Cellular Defense Response

To gain insight into the possible regulatory pathways affected by theC19MC cistron, RNAseq was performed followed by gene set enrichmentanalysis of the 757 genes that were differentially expressed in both759-sgRNA/SAM and 620-sgRNA/SAM compared to control GFP transfectedcells (FDR≤0.10, <−2 and >2-fold), demonstrating that cellular defense,cytokines, interferon and innate immune response to be the most enriched(FIG. 1D). Interferons (IFNs) are known for their potent antiviral andimmunomodulatory activities. Type I IFNs includes 13 IFN-α (IFNAs)subtypes and a single IFN-β (IFNB1) subtype, which can be induced andsecreted from most cell types. They signal through the heterodimericreceptor complex (IFNAR1/2), found in all nucleated cells that activatehundreds of downstream IFN-stimulated genes (ISGs) resulting in a potentsystemic antiviral state. On the other hand, type II IFNs consist ofonly IFN-γ (IFNG), which is induced and secreted predominantly byactivated T cells, natural killer (NK) cells, B cells andantigen-presenting cells (APCs). IFN-γ signals through the IFNγRα andIFNγRβ subunits found on most cell types. Type III IFNs includes IFN-λ1(INFL1), IFN-λ2 (IFNL2), IFN-λ3 (IFNL3) and IFN-λ4 (IFNL4), although thelast is found only in a subset of human population due to asingle-nucleotide polymorphism correcting a frameshift mutation in apseudogene (Prokunina-Olsson, L. et al., Nat Genet 45:164-171 (2013)).Type III IFNs signal through a distinct receptor complex, IFNλR1 andIL10-R2 subunits, found mostly in cells of epithelial origin to activatehundreds of downstream ISGs, similar to type I IFN signaling. Both typeI and III IFNs are key cytokines produced during innate immune responseupon activation of pattern-recognition receptors (PRRs) by pathogensincluding viral, microbial, fungal and parasite infections (Uematsu, S.et al. J Mol Med (Berl) 84:712-725 (2006); Syedbasha, M. et al., FrontImmunol 8:119 (2017)). However, type I IFNs control infectionsystemically whereas type III IFNs are locally induced to controlinfection at barrier surfaces (Wells, A. I. et al., Trends Immunol39:848-858 (2018)). Importantly, human trophoblasts cells constitutivelyrelease type III IFNs, even in the absence of viral infection, and themolecular mechanism that trigger the constitutive expression of IFNs isstill unknown (Wells, A. I. et al., Trends Immunol 39:848-858 (2018);Ander, S. E. et al., Sci Immunol 4 (2019)).

RNAseq analysis show that transcriptional activation of C19MC cistronhighly induced IFNL2 (419.8- and 76-fold in 759- and 620-sgRNA/SAM,respectively), IFNL3 (515- and 104-fold in 759- and 620-sgRNA/SAM,respectively) and numerous IFN-response genes including OAS1. Theincrease in miR-517a, a member of the C19MC, IFNL2 and IFNL3 expressionin AdHEK cells transfected with 579-sgRNA/SAM were confirmed by RT-PCRusing Taqman probes that recognizes both mRNAs due to their >96%sequence homology (FIGS. 1E and 1F). the expression level of IFNLreceptor complex subunits, IFNLR1 and IL10-RB in AdHEK293 were alsomeasured with or without 759-sgRNA CRISPR/SAM, demonstrating comparableCT values of both FNLR1 and IL10-RB (FIG. 1G). Moreover, when increasingamounts (0-4000 μg/mL) of recombinant IFNL1/3 was added to AdHEK293cells media resulted in a dose dependent increase in interferon responsegenes OAS1 and SG15 in manner (FIG. 1H). To further confirm therelationship between C19MC cistron and IFNL2 and IFNL3, the endogenousC19MC cistron was transcriptionally activated using 759-sgRNA/SAM inhuman trophoblast cell line, HTR-8/SVneo cells, that does not expressthe C19MC cistron and found 1600-fold (FIG. 1I) and 99-fold (FIG. 1J)increase in miR-517a and IFNL2/3 expression, respectively. The sameeffects were also found in MCF-7 cells transfected with 759-sgRNA/SAMincreased the expression of C19MC and IFNL2/3 expression (FIGS. 1K and1L). These data confirm that transcriptional activation of the C19MCcistron induces IFNL2/3 expression and its downstream antiviral responsegenes in different cell types, even in the absence of any viralinfections.

C19MC Induces IFN Response in a microRNA Independent Mechanism

To determine whether the increase in IFNL2/3 and the cellular defense inresponse to activation of the C19MC cistron is due to the increase inthe miRNAs of the C19MC cistron, 759-sgRNA/SAM-mediated upregulation ofC19MC was performed in DICER1 knockout HEK293T cells (NoDice 2-20, FIG.2A) (Bogerd, H. P. et al., RNA 20:923-937 (2014)), followed by RT-PCRfor miR-517a and IFNL2/3 where backbone sgRNA (BB-sgRNA) was used ascontrol. As expected, miR-517a was >40 fold induced in 759-sgRNA/SAMtransfected HEK293T cells but not in NoDice 2-20 cells (FIG. 2B).Importantly, the expression levels of IFNL2/3 in NoDice 2-20 cellstransfected with 759-sgRNA/SAM were >100-fold compared to BB-gRNA/Samtransfected cells, very similar to 759-sgRNA/SAM transfected HEK293Tcells (FIG. 2C). Moreover, the levels of IFNL3 in the supernatant ofNoDice 2-20 cells transfected with 759-sgRNA/SAM were 2120 μg/ml,similar to the HEK293T cells transfected with 759-sgRNA/SAM (FIG. 2D).Importantly, the accumulation of pre-miRNA in NoDice 2-20 cells did notinduce INFL2/3 when cells were transfected with 759-sgRNA/SAM orBB-sgRNA/SAM (FIGS. 2B and 10 ). RNAseq was also performed, identifyinggenes that are involved in interferon type III and cellular defense tobe the induced in 759-sgRNA/SAM compared to BB-sgRNA/SAM transfectedcells. These data demonstrate that the increase in the defense and inIFN response after 759-sgRNA/SAM transfection is independent of themature miRNA and suggest that other RNA transcripts present in theC19MC, such as the repetitive Alu elements may be responsible for theinduction of IFNL2/3 and the defense response.

C19MC Induce IFN Response by Increasing Alu RNA

To test whether transcriptional activation of the C19MC cistronincreases the expression of the embedded Alu repeats, primer extensionwas performed using an Alu probe, demonstrating increase in the Alu RNAabundance in both HEK293T and NoDice 2-20 cells transiently transfectedwith 759-sgRNA/SAM compared to BB-sgRNA/SAM (FIG. 3A).

To test whether the increase in IFNL2/3 is due to increase in thetranscription of the Alu elements that are located in the C19MC cistron,the CRISPR/Cas9-SAM technique was used to transcriptionally activateanother large gene CYP19A1, which span over ˜130 kb and is highlyexpressed in syncytiotrophoblast similar to C19MC, but contains only 23Alu elements (FIG. 3B). Two sgRNAs (47.2- and 125.3-sgRNA) were designedthat bind around the 150 bp upstream of the transcription start site.HEK293T cells transfected with 47.2-sgRNA/SAM or 125.3-sgRNA/SAMincreased >500-fold CYP19A1 the expression but did not induce IFNL2/3expression, whereas cells transfected with 759-sgRNA/SAM that activatedC19MC resulted in >100-fold increase in IFNL2/3 (FIGS. 3C and 3D). Thesedata confirm that the increased transcription of the repetitive elementsmay be responsible for the induction in interferon response.

To directly test the effect of Alu RNA on IFNL2/3 expression, and sincethe C19MC cistron contains Alu repeats in both sense and antisensedirections, AluJb-, AluSx-, or AluSz-RNA were in vitro transcribed inboth the forward and reverse direction with 100% pseudouridinesubstitution to reduce the innate immune response to unmodified RNA(Kariko, K. et al., Mol Ther 16:1833-1840 (2008)). HEK293T or HTR8/SVneocells transfected with the sense and antisense AluJb, AluSx or AluSz RNAresulted in increased IFNL2/3 expression compared to cells transfectedwith a control dsDNA, GFP mRNA capped and poly(A)-tailed or a 300 baseGFP RNA fragment transcribed in the sense and antisense (FIGS. 3E and3F). These results further confirm that increased expression of thesense and antisense Alu RNA induce IFN type III in the absence of viralinfection.

C19MC-Mediated Alu RNA Expression Causes Constitutive/FN Response inPluripotent Stem Cells and in the Placenta

In addition to the high level of expression in the placenta, C19MC isalso strongly expressed in pluripotent stem cells. The expression ofIFNL2/3 was examined in iPSCs de-differentiated from human blood(Churko, J. M. et al., Methods Mol Biol 1036:81-88 (2013)),demonstrating high expression of both C19MC miRNA as represented inmiR-517a (FIG. 4A) and IFNL2/3 compared to differentiated normal humanfibroblast (FIG. 4B). C19MC cistron is known to be highly expressed inthe villus trophoblasts and is lost as they differentiate intoextravillus trophoblast. To test whether the expression of C19MC alsoproduce Alu RNA transcripts in situ, in situ hybridization was performedusing probes designed to recognize Alu transcripts on paraffin embeddedterm placental sections pretreated with or without RNaseA. Strongexpression of Alu RNA was found in the villus trophoblasts which wasabolished by RNaseA treatment (FIG. 4C). These data confirm that the Aluprobe recognizes Alu RNA transcripts and not the genomic Alus. Tofurther confirm the co-localization of the Alu transcripts with theC19MC cistron, in situ hybridization was performed using miR-517a/c- orthe Alu probe (a member of the C19MC cistron) on adjacent consecutivelycut paraffin embedded placental sections from first trimester and earlypregnancies. Immunohistochemical staining for cytokeratin and vimentinwere also performed to distinguish between trophoblasts and decidualcells, respectively. Similar to miR-517a/c, Alus were highly expressedin the villus trophoblasts and proliferative trophoblastic cell columnsin anchoring villi and gradually decreased as the trophoblastdifferentiated and invaded the decidua (FIG. 4D). Since Alu repeats andC19MC cistron are specific to primates, in situ hybridization wasperformed with miR-517a/c or Alu probe on paraffin embedded placentalsections from VVT or C19MC transgenic mice (Chang, G. et al., FASEB J31:2760-2770 (2017)), demonstrating that miR-517a/c and Alus were highlyexpressed in the fetal labyrinth and the junctional zone, while thematernal decidual area was negative (FIG. 4C). Taken together, thesedata demonstrate that primates have developed a specific exaptation bywhich constitutive expression of C19MC Alu RNA in pluripotent stem cellsand in the placental villus trophoblasts to induce IFNL2/3 and thecellular defense response to protect the developing fetus before theimmune system is fully developed.

Increase in Alu RNA Induce IFNL Response in Somatic Cells

Alus repeats are the most abundant transposable elements in the humangenome and can be independently transcribed by RNA polymerase III inresponse to various stress conditions. Previous studies have shown thatviral infection increases Alu transcription (Chu, W. M. et al., Mol CellBiol 18:58-68 (1998)), while others showed that viral infection inducesIFNL2/3 expression and interferon response (Kotenko, S. V. et al., NatImmunol 4:69-77 (2003)). It is established that cellular treatment withcycloheximide or heat shock also increase Alu transcription (Chu, W. M.et al., Mol Cell Biol 18:58-68 (1998)). However, the effect of thesetreatments on IFNL2/3 expression was not tested. Here we show that HeLacells treated with cycloheximide or subjected to heat shock alsoincreased IFNL2/3 expression (FIG. 5A). This concomitant increase in Aluand IFNL2/3 expression indicates that Alus may also have been adapted toprime the innate immune response in somatic cells and bolster anti-viraldefenses during cellular stress.

Once considered “junk” DNA or even “parasitic” genes, Alu repeats havebeen found to have functional roles in regulation of gene expressionduring transcription, RNA editing and translation (Hasler, J. et al.,Nucleic Acids Res 34:5491-5497 (2006)). Disclosed herein is a new roleof the Alu repeats in activating type Ill interferon during cellularstress to heighten the anti-viral state. Furthermore, the expression ofthe Alu-rich C19MC cistron in pluripotent stem cells and placentaltrophoblasts drives the constitutive expression of type III interferonand creates a formidable anti-viral barrier to the developing fetus.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

I claim:
 1. A vaccine, comprising in vitro transcribed (IVT) RNAcomprising short interspersed nuclear elements (SINEs) and an antigen ina pharmaceutically acceptable carrier, wherein the IVT RNA comprisespseudouridine and no uridine.
 2. The vaccine of claim 1, wherein theSINE comprises Alu repeats.
 3. The vaccine of claim 1, wherein the SINEcomprises AluJ, AluS, and/or AluY RNA sense strands, antisense strands,or a combination thereof.
 4. A composition comprising: in vitrotranscribed (IVT) RNA comprising short interspersed nuclear elements(SINEs), wherein the IVT RNA comprises pseudouridine and no uridine. 5.The composition of claim 4, wherein the SINE comprises Alu repeats. 6.The composition of claim 4, wherein the SINE comprises AluJ, AluS, orAluY RNA sense strands, antisense strands, or a combination thereof. 7.The composition of claim 4, wherein the IVT RNA comprises a combinationof AluJ, AluS, and AluY RNA sense strands and antisense strands.
 8. Apharmaceutical composition comprising: in vitro transcribed (IVT) RNAcomprising short interspersed nuclear elements (SINEs) and apharmaceutically acceptable carrier, wherein the IVT RNA comprisespseudouridine and no uridine.
 9. The pharmaceutical composition of claim8, wherein the SINE comprises Alu repeats.
 10. The pharmaceuticalcomposition of claim 8, wherein the SINE comprises AluJ, AluS, or AluYRNA sense strands, antisense strands, or a combination thereof.
 11. Thepharmaceutical composition of claim 8, wherein the IVT RNA comprises acombination of AluJ, AluS, and AluY RNA sense strands and antisensestrands.